399. rAAV2 Movement Through the Late and Recycling Endosomes Is Influenced by Titer of Infection

Abstract

Top of pageAbstract The intracellular trafficking of recombinant adeno-associated virus type-2 (rAAV2) to the nucleus is a major barrier for efficient transduction. Using imaging and subcellular fractionation techniques, we evaluated the extent of rAAV2 movement through the late and recycling endosomes following infection in HeLa cells. The intracellular distribution of rAAV2 was monitored using expressed tagged fusion proteins for Rab7 and Rab11 that mark late and recycling endosomes, respectively. Biochemical strategies for evaluating the distribution of rAAV2 genomes in the late and recycling endosome of infected HeLa cells utilized magnetic bead immuno-isolation with ectopically expressed HA-Rab7 or HA-Rab11 endosomal tags. Results from this analysis demonstrated that the majority of AAV2 preferentially trafficked to the Rab-7 positive late endosome at an infection titer of 10 viral genomes/cell. Interestingly, at 100-fold higher titers of infection, rAAV2 movement to the Rab-11 recycling endosome increased approximately 100-fold, becoming the predominant compartment in which the virus accumulated. These results demonstrated that rAAV2 movement to the Rab-7 late endosome saturates at high titers of infection. Intracellular co-localization studies using expressed EGFP-Rab7 or EGFP-Rab11 markers and Cy3-labeled rAAV2 confirmed biochemical studies demonstrating titer-dependent trafficking patterns of rAAV2 through the recycling and late endosomal compartments. Using RNAi to inhibit either the Rab7 or Rab11 pathways, we next sought to determine which compartment might be most efficient at processing virus for functional transduction of a transgene. Results from these studies demonstrated that inhibition of Rab11 more significantly inhibited rAAV2-mediated luciferase gene expression at low titers of infection (10 viral genomes/cell), as compared to inhibition of Rab7. In summary, our findings suggest that rAAV2 trafficking through both the late and recycling endosomes can mediate functional transduction in HeLa cells. Although rAAV2 movement to the recycling endosome appears to be less efficient at low MOIs, processing of the virus through this compartment may be more effective at cellular transduction (i.e., expression of a virally encoded transgene) than through the late endosome. These findings suggest that strategies to shunt viral movement from the late to the recycling endosome may be effective at increasing the efficacy of viral transduction for gene therapy.